Proposed is a plan of research designed to determine the three-dimensional fine structure of mammalian and Drosophila melanogaster diploid mitotic chromosomes in a variety of biologically well-defined functional states. The long-range goal is to understand the structural complexities that underlie DNA condensation and its organization into higher-order structures that can support as well as modulate transcriptional activity, and that change throughout the cell cycle. This is a continuation of a well established research program that has made dramatic progress in the area of three-dimensional data collection methods for both the light and electron microscopes. The technological research has been driven by the need for tools suitable for examination of diploid chromosome structure and organization. Initial studies focus on understanding the structure of the 30nm fiber and how this fiber is packaged into the 130nm higher-order structure. Previous work had shown this 130nm fiber to be a dominant higher-order structural motif that persists throughout the cell cycle, but that is most clearly visualized in telophase. Intermediate Voltage Electron Microscope Tomography (IVEM-T) is being used to provide high-resolution (50-75 Angstroms) three-dimensional reconstructions from thick sections of embedded and stained chromosomes. The structural analyses rely extensively on recently developed computer methods for three-dimensional image reconstruction, enhancement, display and interpretation. These methods are particularly powerful and require neither crystalline specimens nor internal symmetry. Our current studies on HeLa telophase chromosomes will be extended to Drosophila embryonic and imaginal disk chromosomes and mouse lymphocyte chromosomes with experiments designed to target reconstructions to a uniquely defined region on a particular chromosome. Furthermore, these studies will be enlarged to consider a variety of cell cycle stages. Work will continue on the development of general methods designed to make IVEM-T considerably easier to accomplish. Our goal is to significantly broaden the accessibility of this powerful method for general use in cell biology.